The energetic foundation of cellular activity and its regulation relies on a string of oxidoreductase proteins in biological oxidative and reductive metabolism and respiratory membrane energy conversion. The mechanisms of many catalytic sites of substrate oxidation-reduction and energy conversion, particularly in mitochondrial respiration, have proven to be difficult to access experimentally due to natural complexity and fragility. They remain poorly understood. Our proposal aims to reveal the natural engineering of the redox-coupled proton exchange and transfer operating at these sites of multi-electron cofactor and substrate oxidation-reduction. Our approach builds on our engineering guidelines for protein electron tunneling, strengthened in the last grant period, to inform the de novo design and assembly of simple and robust alpha-helical proteins intended to serve as protein-based models, maquettes, for multi-electron catalysis. Maquettes will be designed to provide the simplest water-soluble or trans-membrane structures that can capture the functional properties of natural redox centers. Their simplicity and adaptability allow us to investigate catalytic functional problems that remain unsolved in the respiratory chain. Maquettes will be activated with light and electrometric methods in solution and on electrodes to dissect step-by-step the thermodynamics and kinetics of electron transfer and proton exchange. Maquettes will incorporate all key 2-electron, multi-proton cofactors/substrates quinone, nicotinamide and flavin, and the 2-and 4-electron substrate O2. We are positioned to focus on the problem of reversible energy conversion catalysis of hydroquinone-quinone in the Qo site of the cytochrome bc1 aiming to determine the mechanistic root of medically harmful short-circuits and radical generation. We will extend our maquette creation of a stable O2 ferrous heme state, to examine 2-and 4-electron O2 reduction and the physiologically important 2-electron chemistries of NO and H2O2. With stable and adaptable maquettes, we can exploit physiological chemistry in the development of nanoscale devices. The breakdown of food by oxygen respiration in humans produces all the energy needed for a healthy life and is a central part of cellular regulation. It is normal that there is a steady slow release of oxygen radicals that over time cause cellular damage, aging and disease. Under stress, and during many surgical procedures bursts of radicals can accelerate these deleterious processes. The research of this grant describes a new way to understand the processes underlying healthy oxidative energy supply and control as well as deleterious radical generation. With progress we will be better predict and track the onset of disease and act to slow it or reverse it. [unreadable] [unreadable] [unreadable]